Pattern specific melanin synthesis and DOPA decarboxylase activity in a butterfly wing of Precis coenia Hübner

Inserr Biochem. M&c.

09651748(94)ooo40-9

Biol. Vol. 25, NO. I, pp. 73-82, 1995

Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0965-1748195 $9.50 + 0.00

Pattern Specific Melanin Synthesis and DOPA Decarboxylase Activity in a Butterfly Wing of Precis coenia Hiibner P. B. KOCH,

N. KAUFMANN

Received 1 September 1993; revised and accepted 11 May 1994

Black pigment extracted from wings of Precis coeniu was identified as melanin by solubility characteristics and incorporation of radiolabeled melanin precursors. Different colours in wing scales appeared successively, starting with white followed by red, black and grey pigments. At corresponding times (‘4C]-tyrosine and [14C1-3,4dihydroxyphenylalanine injected into pupae were incorporated most intensively into black and grey scales. By contrast, red scales were labeled by 3,4dihydroxyphenylalanine (DOPA) and, to a lesser extent, by tyrosine when injected at the time of red pigment synthesis. Labeled /l -alanine, a component of the sclerotizing agent N-/l -alanyldopamine, was incorporated into all scales with the exception of intensely black scales. It is discussed whether the mechanical stability of black scales may be due to melanin itself whereas the stability of all other scales may be due to N-/I -alanyldopamine. Amounts of extractable melanin increased during visible pigment formation in black and grey scales. When (‘4C]-tyrosine was offered to isolated wings in Grace’s medium before melanization, 93% of incorporated activity was found in the hydrolysable fraction and only 7% in the melanin fraction. However, when supplied during intensive melanin synthesis up to 70% was incorporated into melanin. The same incorporation pattern resulted after injection into intact developing pupae. Incorporation of [‘4C]-tyrosine into melanin began earlier, and was higher in presumptive black scales than in grey scales. Incorporation into white scales was always low. DOPA decarboxylase (DDC) activity in whole wings was low during the pupal stage but increased at the time when scale melanization started. DDC activity in different coloured wing pieces increased first in presumptive black pattern elements, the eyespots. DDC activity in the eyespots was 3.5 times higher than in presumptive white parts of the wing; it was intermediate in grey areas. This demonstrates that wing colour pattern as well as colouration intensity are related to selective activity of DDC in time and different locations resulting in different amounts of melanin in grey and black scales. Melanin Tyrosine coenia Nymphalidae

Dihydroxyphenylalanine Lepidoptera

j? -alanine

INTRODUCTION

Allgemeine

Zoologie,

Universitiit

Ulm,

Colour

pattern

Precis

butterfly species investigated so far, different wing pigments are synthesized in an invariable time course starting with white (pteridines), followed by red (ommatins) and black and grey (melanins) (Nijhout, 1980b, 1991, Koch, 1992). Possibly this is a general feature in all Lepidoptera. Pigment synthesis in scale cells has been investigated in vitro for pteridines (Descimon, 1975), ommatins (Koch, 1991) and melanins (Nijhout, 1980b), indirectly demonstrating the presence of enzymes required for the respective pigments. Precursors of cuticular melanin synthesis have been studied in several insects, e.g. in larval integument of Manduca sexta (Hiruma and Riddiford, 1984; Hori et al., 1984), in differently pigmented strains of a beetle, Tribolium castaneum (Kramer et al., 1984; Roseland et al., 1987)

A butterfly wing colour pattern is made up of an array of differently coloured scales. Scales may contain different kinds of pigments, e.g. melanins, pteridines and ommochromes (Kayser, 1985; Nijhout, 1985). Within a single scale the colour appears homogenously. Differently coloured scales may contain different amounts of a specific pigment or eventually mixtures of different pigments (Nijhout and Koch, 1991). The latter was originally suggested for different forms of melanins which can appear from red and brown to black depending on the melanin precursor (Nijhout, 1980b). In all Abteilung Allee

Dopadecarboxylase

Albert-Einstein-

11, D-89069 Ulm. Germany. 13

74

P. B. KOCH and N. KAUFMANN

and in the order of Dictyoptera (Czapla et al., 1990). However, investigations on melanins in wing scales are rare. In Precis coenia wing melanization has been studied in vitro by adding different precursors to the incubation medium (Nijhout, 1980b). Pattern specific incorporation of [‘4C]-tyrosine was studied by injections into pupae and subsequent autoradiography (Nijhout and Koch, 1991). In the present paper the time course of incorporation of radiolabeled [‘4C]-tyrosine is investigated after short term in vitro supply to isolated wings. Two alternative models may account for the regulation of pattern specific pigment synthesis: (A) All pigment precursors have access to all scales and selective enzyme activity is regulating the colour. (B) A variety of pigment forming enzymes are present in every scale and selective uptake of precursors is regulated (Nijhout, 1980b; Koch, 1992). However, both mechanisms may be involved. Of the enzymes involved, phenoloxidase and DOPA decarboxylase (DDC) were shown to have important roles in cuticular melanin formation in M. sexta larvae (Hiruma et al., 1985; Hiruma and Riddiford, 1990, 1993). Therefore, DDC may have a role in synthesis of butterBy scale melanin as well. In the present paper we investigated: (I) in vivo and in vitro incorporation of melanin precursors into different elements of the colour pattern; (II) incorporation of tyrosine, DOPA and ,!?-alanine into melanin; (III) differentiated incorporation of radiolabel into differently coloured portions of the wing; and (IV) activity of DDC during wing development and in different elements of the colour pattern. The results support the role of differential enzyme activity in colour pattern formation as was assumed in model (A).

MATERIALS AND METHODS Animals and wing development

The North American Buckeye, Precis coenia Hiibner (Nymphalidae), was reared on semiartificial diet as described previously (Nijhout, 1980,a). Animals were kept under long-day conditions (16L : 8D) at 25.5”C with lights on at 07 : 00 and lights off at 23 : 00. Pupal development including the onset of wing colouration is gated by photoperiod (Nijhout, 1980a). Only those pupae which developed within 7 days were used for experiments. Time course of synthesis of different pigments has been described by Nijhout (1980b) and is important for times of incorporation of pigment precursors (see Fig. 1A). Chemicals and autoradiography

L[U-14C]-tyrosine (specific activity 463 mCi/mmol), L-[~-‘~C]-DOPA (12 mCi/mmol) were obtained from Amersham and [l-14C]-/I-alanine (53 mCi/mmol) from New England Nuclear. All other chemicals of p. a. quality were purchased from Merck or Sigma. Radiolabeled precursors were dissolved in Grace’s medium and volumes of 10~1 were injected laterally behind the 5th abdominal segment of the pupa. Doses of

0.1 or 0.2 PCi L-[U-‘4C]-tyrosine and 0.5 PCi L-[~-‘~C]DOPA or [l-‘4C]-/?-alanine were injected at different times of pupal development. Scales were removed from the wings after adult emergence (Koch, 1991) and exposed to x-ray film (Kodak x-omat AR) for autoradiography. Six forewings from pupae injected with labeled precursurs on the evening of day 6 were used for melanin extraction. Incubation of isolated wings

Wings were dissected from pupae under sterile conditions as described previously (Koch, 1991). They were incubated in volumes of 1 ml Grace’s medium (Gibco) at 25.5”C and shaken at 100 rounds/min in 24-well tissue culture plates (Costar). The plates were placed in a plastic box supplied with oxygene (25 ml/min). For incorporation studies, wings were incubated in medium initially prepared without tyrosine and then supplied with 0.1 pCi/ml of [‘4C]-tyrosine or [‘4C]-DOPA for 24 h followed by two washes in radiolabel-free Grace’s medium for 3 h. In order to study the time course of incorporation wings at different developmental stages were incubated for 5 h (pulse) in Grace’s medium additionally containing 0.5 pCi/ml [‘4C]-tyrosine. Subsequently, wings were washed two times and incubated in radiolabel-free medium (19 h chase). Incorporation of B-alanine was studied in medium containing 1 pCi/ml [‘4C]-/I-alanine. Wings were dried between blotting paper and exposed for autoradiography. Extraction and quan@cation

of melanin

Melanin was extracted according to Hackman and Goldberg (1971) modified after Maisch and Biickmann (1987). For determination of the extinction coefficient, 25 adult forewings were washed in 15 ml acetone (24 h) and hydrolyzed twice in 10 ml 6 M HCl at 90°C (19 h). After centrifugation, melanin was extracted from the remaining wing fragments three times with 15 ml 10% NaOH at 90°C (6 h). Pooled NaOH-extracts were cleared by centrifugation and melanin was precipitated with 15 ml 32% HCl. The melanin pellet was washed with distilled water and pure ethanol, dried and weighed. Melanin was dissolved in 10% NaOH in different concentrations. The mean extinction coefficient at 470 nm was 2.7 (litre x g-’ x cm-‘). Melanin contents in single forewings were measured after extraction in volumes of 1 ml. Incorporation of tyrosine into melanin

Radiolabeled [‘4C]-tyrosine (0.2 PCi) was injected into pupae of different ages. Following adult emergence, melanin was extracted from samples containing four forewings. After washes with acetone and pure ethanol wings were hydrolyzed in 3 ml of 6 M HCl (3 x 24 h at 9OC). Acid soluble fractions were pooled, aliquots were neutralized with 6 M NaOH and radioactivity was measured by liquid scintillation counting (LSC) in a Packard Tricarb C. Melanin extracted with 10% NaOH (3 x 24 h at 90°C) was determined photometrically and

PATTERN

SPECIFIC

MELANIN

SYNTHESIS

radioactivity was measured in LX. Remaining wing fragments still containing black pigment were sucked onto glass fibre filters (Whatman GF/A, 2.4 cm), washed with water, 70% ethanol and pure ethanol and radioactivity was counted by submersing the filters under a 15 ml scintillator. In vitro incorporation of [‘4C]-tyrosine into melanin was measured after pulse-chase incubation (see above). In order to remove regions which had formed melanin as a consequence of injury, the basal parts of the wings were cut off. Samples of 4 forewings were pooled for melanin extraction and radioactivity were determined as described above. Pattern specific incorporation of [‘4C]-tyrosine into melanin of different coloured parts of the wing was

AND

DOPA

DECARBOXYLASE

measured in the black eyespot, the white band and an intermediate coloured region (see Fig. 8). Following pulse-chase incubation, 16 circles were punched out from each region (of 16 wings) by means of a stainless steel stamp with 1 mm inner diameter. These samples were hydrolyzed in 6 M HCI for 48 h at 90°C. The wing fragments containing melanin were sucked onto glass fibre filters and radioactivity was measured as described above. Measurement

of DOPA decarbox_ylase actiuit,y

DOPA decarboxylase (DDC) was extracted from freshly dissected forewings or from wing pieces by homogenization in a 1 ml all-glass homogenizer (Wheaton-Duall) in homogenization buffer (Hiruma

d5e

d5 20~00

d8 8~00

d8 12~00

15

d6 1800

d6

20~00

d7 8~00

time

FIGURE 1. (A) Right pupal forewings at different developmental stages before incubation (top) and left wings from the same specimen after 24 h incubation in Grace’s medium (below). On day 5 at 2090 hours (d5e) white pigments appeared. On day 6 at 8:OO (d6m) red pigment synthesis started and was continued to day 6 at 20:00 (d6e) when black eyespot already appeared. On day 7 morning (d7m) wing pigmentation appeared to be complete about 3 h before adult emergence. Black bar indicates natural length of a pupal wing. (B) Amounts of extractable melanin from pupal wings during visible pigment formation. Bars represent SEM from 3 determinations.

76

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and N. KAUFMANN

TABLE 1. Percentage of radioactivity in 6 M HCl hydrolysate, melanin extract in NaOH and in remaining wing fragments recovered from adult wings after injection of different radiolabeled precursors on day 6 evening. Activity in NaOH extract and in remaining wing fragments are combined to “total melanin”. Mean values from two determinations with SEM Percentage HClhydrolysate

Precursor [‘%I-tyrosine [‘%I-DOPA [‘4C]-r?-alanine

NaOHextract

49.5 + 1.8 67.4 + 1.5 92.5 + 0.2

36.9 + 1.5 27.5 + 0.9 7.0 + 0.2

et al., 1985). Samples of one forewing were homogenized

in 500~1. Samples of 20-26 wing pieces (Fig. 8) were dissected in ice cold Grace’s medium using microscissors and then immediately homogenized in 300 ,ul buffer. After two centrifugation steps (16,000g for 15 min), clear supernatants were stored at -20°C until enzyme assay. DDC activity was measured by the radiometric assay of McCamman et al. (1972) incubating 10 ~1 of the sample with 30 ~1 of reaction mixture (three replicates) for 30 min in 37°C. Protein contents in 100 ~1 of the samples (two replicates) were determined photometrically (Appenroth and Augsten, 1987) immediately after the enzyme assay and were used as a correction factor for different sizes of wings and wing pieces. DDC activity is expressed as relative enzyme activity in desintegrations per minute (d.p.m.) per pg protein. In each series of DDC assay a standard sample from Drosophila white prepupae with high DDC activity (Kraminsky et al., 1980) was used as a control. RESULTS

Melanin

deposition

radioactivity Wing fragments 13.6 + 0.4 5.1 + 0.5 0.5+0

Total melanin

Total activity

50.5 + 1.8 32.6 + 1.5 7.5 + 0.2

100 100 100

some black pigment, presumably the result of residues of melanin. [‘4C]-tyrosine injected into pupae on day 6 evening was incorporated into wing scales (Fig. 3D). Acid hydrolysis of adult wings yielded 49.5% of total incorporated radioactivity, 36.9% was extracted by NaOH and 13.6% remained in the wing fragments. Therefore, about 50% of radioactivity was incorporated into melanin (Table 1). Injected [14C]-DOPA was incorporated to a lesser extent into melanin, namely 32.6%. By contrast, only 7% of radioactivity from injected [‘4C]-/?-alanine was extracted by NaOH and almost nothing (0.5 %) remained in the wing fragments (Table 1). [‘4C]-tyrosine injected into pupae of different ages was incorporated into wing melanin in different amounts. Injection prior to melanization resulted in high radioactivity in the acid soluble fraction and low activity in the melanin fraction. Injection on day 6 evening before intensive melanization led to the highest rate of incorporation into melanin (Fig. 2). Pattern specljic incorporation into intact pupae

during wing development

Melanin synthesis started during day 6 of pupal development when white and red pigments are visible. Melanization appeared first in the presumptive black eyespot followed by black stripes next to the red bands and then started in dark-grey scales (Figs. IA, 3A). Grey to black pattern elements continued to become darker during the night from day 6 to day 7. Extractable melanin increased in the wings over this period of successive melanization (Fig. 1B). Melanization also occurred in isolated wings cultured in Grace’s medium containing tyrosine as the only melanin precursor. Different completion of the colour pattern was reached depending on the stage when a 24-h incubation was initiated (Fig. 1A). No melanization occurred at all when the medium lacked tyrosine. Melanization did not occur when the wings were not shaken in the medium because of lack of oxygen.

of precursors

after injection

Injection of [‘4C]-tyrosine in l-5 days old pupae caused a weak labeling of black scales (Figs 3A, B).

L

I

\

. 24 I

12

24

45

12

24

dd

fl

Time (hi

Incorporation

of precursors

into extractable

melanin

Red and dull brown pigments were removed from the scales by hydrolysis with 6 M HCI. Grey and black pigment withstood this treatment (Fig. 3H). Following this treatment, black pigment was extracted with 10% NaOH and the remaining wing fragments still contained

FIGURE fraction fragments ent aged melanin melanin

2. Percentage of extracted radioactivity in acid soluble (open squares), melanin fraction (open triangles) and wing (closed triangles) after injection of [‘%I-tyrosine into differpupae and extraction from adult wings. Radioactivity of total (closed circles) as the sum of extractable and fragment bound increased during wing pigmentation. Bars represent SEM from 2 determinations.

PATTERN

SPECIFIC

MELANIN

SYNTHESIS

AND

DOPA

DECARBOXYLASE

FIGURE 3. Dorsal fore- and hindwing of Precis coeniu (A) with black eyespot (b), white band (w) and red proximal bands (r). Autoradiograms show labeling of scales after injection of [‘%J]-tyrosine into pupae on day 2 (B), on day 6 morning (C) and day 6 evening (D) and labeling from [‘4C]-DOPA when injected on day 6 morning (E) and day 6 evening (F) and labeling from [“Cl-fi-alanine when injected at any time of pupal stage (G). Radioactivity is represented by light areas. Hydrolysis in 6 M HCl for 3 days at 40°C removed red and dull brown colours from the wing, whereas black and grey pigments withstand this treatment (H bottom) compared to an untreated wing (H top). All wings in natural size.

Injection on day 6 morning during red pigment formation caused weak labeling of red and black scales (Fig. 3C). Only 12 hours later, on day 6 evening, [‘4C]-tyrosine produced intensive labeling of black and grey scales (Fig. 3D). [‘4C]-DOPA was incorporated into red scales when injected on day 6 morning (Fig. 3E) and into red, grey and black scales when injected on day 6 evening (Fig. 3F). Earlier injections did not label scales. Injection of [‘4C]-fi-alanine resulted in some kind of reverse labeling. Strikingly, black and dark grey scales were not labeled. Grey scales were weakly labeled, but white, red and dull brown scales were all intensively labeled when injected on day 6 evening (Fig. 3G) or earlier. Pattern specljic incorporation of precursors wings in tlitro

by isolated

Isolated wings incubated in Grace’s medium incorporated both, [‘4C]-tyrosine and [‘4C]-DOPA in a pattern specific manner (Fig. 4A, B). Pulse-chase labeling experiments with 0.5 PCi [‘4C]-tyrosine per ml medium for 5 h (pulse) were started every 8 h beginning on day 5 at 24: 00 when white pigments are visible (compare Fig. 1A). At this first stage the whole wing was weakly labeled (Fig. 4D). Incubation started when red pigments are just becoming visible (day 6, 8:00) resulted in labeling of white and red scales. Red scales, black eyespot and some other prospective black scales were

labeled at the time when melanization of the black eyespot begins (day 6, 16:OO). Later on, starting on day 6 midnight or on day 7 morning only 2-4 h before adult emergence, [‘4C]-tyrosine intensively labeled black and grey scales (Fig. 4D). This time course demonstrates that tyrosine incorporation and melanin synthesis do not only begin earlier but also last longer in the black eyespot than in dark grey and grey portions of the wing. [‘4C]-p-alanine was incorporated by isolated wings in the same manner as after injection into intact pupae (Fig. 4C). Incorporation of tyrosine into melanin by isolated wings Following pulse-chase labeling with 0.5 PCi [‘“Cltyrosine (see Fig. 4D), the percentage of radioactivity incorporated into the hydrolysis fraction was high before pigment synthesis started and then decreased with the beginning of melanin synthesis. By contrast, radioactivity in the melanin fractions, NaOH-extract and remaining wing fragments, increased during visible melanization (Fig. 5). This demonstrates that labeling of grey and black scales in autoradiography is due to incorporation into melanin. Pattern specific incorporation of [‘4C]-tyrosine into melanin was measured by cutting out samples from black eyespots, white bands and intermediate coloured parts of the wings after pulse-chase labeling. Radioactivity in white wing pieces was always low, regardless of the stage when incubation began (Fig. 6, white

78

P. B. KOCH

and N. KAUFMANN

FIGURE 4. Autoradiograms of pupal forewings after incubation with [i4C]-tyrosine (A), medium starting on day 6 morning. Autoradiogram after incubation with [‘%Z]-/I-alanine (right) wing sides after incubations started on day 6 morning (top) and day 6 evening compare to Fig. 1. (D) Autoradiograms of dorsal forewings after 5 h pulse-chase incubation 5 at 2430 (5524); day 6 at 8:00 (68); day 6 at 1690 (616); day 6 at 24:00 (6-24)

columns). Incorporation into melanin of eyespots started on day 6, between 8: 00 and 16:OO when visible melanization commenced. After 8 h, radioactivity increased also in intermediate (grey and dull brown)

[14C]-DOPA (B) for 24 h in Grace’s (C) shows dorsal (left) and ventral (below). For natural pigmentation with [“‘Cl-tyrosine started on day and on day 7 at 7:00 (7 17).

coloured portions of the wing (Fig. 6). Therefore, melanin synthesis starts earlier and lasts longer in prospective black than in intermediate coloured portions. 10,000-

7500. .E i% 5 E

sooo-

.G

E 8

12

24

12

24

d2

I

d6

t

12 67

2600-

24 I

Time (h) FIGURE 5. Percentage of extracted radioactivity in acid soluble fractions (open squares) and total melanin (closed circles) after 5 h pulse-chase incubations of isolated wings with [‘%I-tyrosine. Symbols as in Fig. 2. Times of samples correspond to autoradiograms in Fig. 4D.

Od5 16:OO

d8 24:OO

d7 1:oo

FIGURE 6. Radioactivity of white (white columns), intermediate coloured (dotted) and black eyespot (crossed) wing pieces after acid hydrolysis from wings incubated in 5 h pulse-chase experiments as in Fig. 4D. Radioactivity represents activity in melanin of one sample pooled from 16 forewings each.

PATTERN

SPECIFIC

MELANIN

SYNTHESIS

AND

DOPA

19

DECARBOXYLASE

* 3000

.E a, p 0.

-

2000-

7 E D”

lOOO-

OI

1

t

I

I

1

2

I

3

P

1

I

4

5

Days

I

6

’

t7

8

’

I

’

Q

A

FIGURE 7. Activity of DOPA decarboxylase in whole wings (solid line) and in black eyespots (broken line) at different times of pupal development. Time sequence of pigment synthesis of white (striped bar), red (dotted bar) and black and grey pigments (black bar) are indicated. P = larval-pupal moult; A = adult emergence. Mean values with standard error of means from three (eyespots) and eight (whole wings) determinations.

DOPA decarboxylase

activity

DDC activity in whole wings decreased during the two days after larval-pupal moult reaching undetectable levels. Thereafter, DDC activity increased dramatically

during day 6 when melanin synthesis begins in grey and black scales (Fig. 7). DDC activity in presumptive black eyespots, which are the first to start melanization, increased earlier and to higher levels (Fig. 7, broken line) than in whole wings.

07

8

8

A

Days FIGURE 8. (A) Schematic representation of white (W), intermediate/grey (G) and black (S) wing pieces which were dissected for measurement of DDC activity. (B) DDC activity in black (solid line), intermediate (dotted) and white (broken line) wing pieces during late pupal to early adult development. Mean values with standard error of means from three determinations. Symbols as in Fig. 7.

80

P. B.

KOCH

and N. KAUFMANN

DDC activity determined in black, grey and white coloured areas (Fig. 8A) showed different time profiles. It increased earlier and to higher levels in presumptive black eyespots, reaching its peak about 10 h before adult emergence (Fig. 8B). Activity increased slowly in white areas reaching its maximum after adult emergence. At the maximum of DDC activity in black eyespots it was 3.5 times higher than in white areas. One day after adult emergence enzyme activities in the different wing parts were about the same and subsequently decreased to lower levels (Fig. 8B). DISCUSSION Melanin in butterfly scales

Dark brown to black pigments in insects are often presumed to be melanin though, in most instances no chemical data are available (Kayser, 1985). Darkening of insect cuticle may be caused by tanning as well as by melanin formation. Tanning occurs in the course of cuticle sclerotization. It requires the same precursor, tyrosine, as melanin formation (Andersen, 1985, 1990; Kayser, 1985). Tyrosine is metabolized to dopamine which is a precursor not only of sclerotization agents N-acetyldopamine (NADA) and N-/?-alanyldopamine (NBAD) (Karlson and Sekeris, 1962; Hopkins et al., 1982; Andersen, 1990) but also of insect melanins (Hori et al., 1984; Roseland et al., 1987; Koch, 1994). Black and various degrees of red to brown pigments in butterfly scales have been speculated to be melanins due to colour formation in isolated wings caused by different precursors (Nijhout, 1980b). In the present paper, red pigments were shown to be hydrolysable with hydrochloric acid whereas grey and black pigments withstand this treatment (Fig. 3H). Following this hydrolysis, grey and black pigments in scales are dissolved in alkaline solution which is a feature of melanins (Maisch and Biickmann, 1987; Hiruma and Riddiford, 1993). Additionally, the incorporation of radiolabeled tyrosine and DOPA into extractable black pigment demonstrates its nature as melanin. Furthermore, we have shown that red pigments in P. coenia do not belong to melanins but derive from tryptophan (Nijhout and Koch, 1991; Koch, 1993) and contain xanthommatin (P. B. Koch, unpublished). Precursors of melanin synthesis in wings

Radiolabeled tyrosine was incorporated into melanin after injection into whole pupae (Fig. 2) as well as after supply to isolated wings in culture medium (Fig. 5). Tyrosine as the only melanin precursor in Grace’s medium allows the development of a perfect pigment pattern in isolated wings and therefore is sufficient for melanin synthesis (Fig. 1A). a-Methyl-p-tyrosine, a competitor for tyrosine hydroxylase (Nagatsu et al., 1964), and m-hydroxy-benzylhydrazine, an inhibitor of DDC, inhibited melanin synthesis in isolated Precis wings. At the same time melanization which is due to injury by wing cuts was not inhibited (Koch, 1994). This

demonstrates that different enzymes are involved in synthesis of scale melanin and immunological melanization, respectively. In scales both, tyrosine and DOPA, are used as precursors by isolated wings in vitro (Figs 4A, B). Furthermore, activity of DDC in different coloured parts of the wing (see below) indicate that the conversion of DOPA to dopamine occurs in the wing pattern specifically. All these results demonstrate that the complete pathway from tyrosine via DOPA and dopamine to melanin occurs in the wings of P. coenia. A similar situation was found in M. sexta cuticle melanin synthesis. Tyrosine occurs in the hemolymph during periods of sclerotization and melanization (Ahmed et al., 1983; Kramer and Hopkins, 1987), but is not the direct substrate in cuticlar melanin granules (Hiruma et al., 1985). Tyrosine is converted to DOPA and dopamine in hemolymph and fat body and furthermore, decarboxylation of DOPA to dopamine in the epidermis is most important for cuticular melanin synthesis (Hori et al., 1984; Hiruma et al., 1985; Hiruma and Riddiford, 1993). The sequence of pigment formation in d@erently coloured scales

There is a sequence of pigment formation in butterfly wings starting with white and followed by red, black and grey (Nijhout, 1980b, 1991; Koch 1992). Supply of precursors [‘4C]-tyrosine or [‘4C]-DOPA to pupae or isolated wings at the time of melanization led to the most intensive label of black scales (Figs 3 and 4). This is clearly due to incorporation into melanin (Figs 2 and 5). Visible pigment formation starts earlier in prospective black scales than in grey scales (Fig. 1A) and pulse-chase labeling demonstrates that melanin synthesis lasts longer in prospective black scales than in grey scales (Figs 4D, 6). Apparently black scales receive more tyrosine and synthesize more melanin during a longer period of synthesis than grey scales. However, labeled tyrosine and DOPA are also incorporated into scales others than black and grey. Precursors supplied at the time before any pigment synthesis labeled the whole wing with a comparatively low intensity. Apparently, at this time they are incorporated into other components of the cuticle than melanin. This is consistent with the result that radioactivity was predominantly found in the acid soluble fraction and not in the melanin (Figs 2 and 5). When supplied at the time of white and red pigment synthesis, both tyrosine and DOPA were specifically incorporated into white and, with a higher intensity, into red scales. This is most striking marked after supply of [‘4C]-tyrosine in vitro starting on day 6 morning (Fig. 4D, 6-8). What is the function of tyrosine and DOPA in scales which do not contain melanin? Red wing pieces were shown to contain xanthommatin derived from tryptophan by extraction with HCl/methanol (Koch, 1993 and unpublished results) and [‘4C]-tyrosine is not incorporated into melanin during the period of white and red pigment synthesis. Two possibilities may account for the intense label of red scales: the Precis red pigment is an

PATTERN

SPECIFIC

MELANIN

SYNTHESIS

AND

DOPA

DECARBOXYLASE

81

unknown pigment containing xanthommatin and a longest and highest DDC activity develop into the darkest, intensely black scales. This result provides the derivate of tyrosine metabolism or tyrosine and metabolites are involved in sclerotization processes in red first biochemical evidence that butterfly colour patterns scales but not in pigmentation. The second possibility is depend on differentiated enzyme activity in the scales. considered more likely. Scales seem to gain their mechanical stiffness during the time of pigment synthesis. This CONCLUSIONS can be concluded from the succession of a relief pattern which was described in P. coenia. Scales remain in a Grey and black scales contain melanin as shown by upright position after air drying the wings but only when incorporation of precursors and solubility properties of pigmentation of respective scales is completed (Nijhout, the pigment. The amounts of melanin being higher in 1980b). It is reasonable to suppose that this coincides intensely black scales than in grey scales are achieved by with some kind of sclerotization process in the scales longer or shorter duration of melanin synthesis respectwhich use tyrosine derivates as sclerotization agents, e.g. ively. Pattern specific melanin synthesis as well as variNBAD and NADA (Anderson, 1990; Umebachi, 1990). ation in the amount of melanin per scale in butterfly fi-Alanine, a component of NBAD, is known to be wings are the result of selective activity of DDC in the incorporated into butterfly wing scales but especially not scales. into intensely black scales (Umebachi, 1990). The result in Precis confirms this distribution of /I-alanine in differently coloured scales after injection into pupae. REFERENCES Additionally, in vitro experiments reveal that /I-alanine is incorporated directly by isolated wings at the time of Ahmed R. F., Hopkins T. L. and Kramer K. J. (1983) Tyrosine and tyrosine glucoside titres in whole animals and tissues during develpigment synthesis and acquisition of mechanical stiffness Munduca sexta (L.). Insect opment of the tobacco homworm (Fig. 4C). Interestingly, intensely black scales acquire Biochem. 13, 369-314. their mechanical stiffness without incorporation of fi- Andersen S. 0. (1985) Sclerotization and tanning of the cuticle. In Comprehensive Insect Physiology Biochemistry and Pharmacology alanine and therefore, sclerotizing agents in intensely (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 3, pp. 59-74. black scales must be other than NBAD. As an alternaPergamon Press, Oxford. tive hypothesis, black scales may gain their mechanical Andersen S. 0. (1990) Sclerotization of insect cuticle. In: Molting und stability through an interaction with melanin. In black Metamorphosis (Edited by Ohnishi E. and Ishizaki H.), Japan scales the cuticle (scale architecture) may be stabilized by Scientific Press/Springer-Verlag, Tokyo/Berlin. the polymeric molecule of melanin. By contrast, in Appenroth K.-J. and Augsten H. (1987) An improvement of the protein determination in plant tissues with the dye-binding method differently coloured scales tyrosine may be precusor of according to Bradford. Biochem. Physiol. Pfianzen 182, 85-89. sclerotization agents which stabilize the cuticle by cross- Czapla T. H., Hopkins T. L. and Kramer K. J. (1990) Catecholamins linking of proteins. This alternative use of tyrosine has . and related o-diphenols in cockroach hemolymph and cuticle during been proposed for differently coloured pupal cuticle in sclerotization and melanization: comparative studies on the order Dictyoptera. J. Comp. Physiol. 16OB, 175-l 81. the nymphalid Znachis io (Maisch, 1985). DOPA decarboxylase synthesis

and

pattern

spec@ic

melanin

Synthesis of different pigments in the colour pattern of butterflies must be regulated in the scales. The most probable hypothesis is that different pigments are synthesized due to selective enzyme synthesis as a product of selective gene expression in the scales (Nijhout, 1980b; Koch, 1991, 1992). The enzyme DOPA decarboxylase (DDC) converting DOPA to dopamine has a regulative role in cuticular melanin synthesis in Manduca (Hiruma et al., 1985; Hiruma and Riddiford, 1990, 1993). DDC activity increased in the wings of P. coenia at the time of melanin synthesis (Fig. 7). Furthermore, DDC activity measured in differently coloured wing areas increased earlier and to higher levels in prospective black eyespots than in prospective grey or white wing pieces. This spatial time pattern of DDC activity exactly corresponds to visible melanization of the scales as well as to incorporation of [‘4C]-tyrosine into melanin. The results suggest that DDC is involved in selective melanin synthesis and also causes different intensities of melanin synthesis leading to more (black) or less melanin (grey) in the scales. Those scales which show the earliest,

Descimon H. (1975) Biology of pigmentation in pieridae butterflies. In Chemistry and Biology of Preridines (Edited by Pfleiderer W.), pp. 8055840, W. de Gruyter., Berlin. Hackman R. H. and Goldberg M. (1971) Microchemical detection of melanins. Analyt. Biochem. 41, 279-285. Hiruma K. and Riddiford L.M. (1984) Regulation of melanization of tobacco hornworm larval cuticle in vitro. J. Exp. Zoo/. 230, 3933403. Hiruma K. and Riddiford L. M. (1990) Regulation of dopa decarboxylase gene expression in the larval epidermis of the tobacco hornworm by 20-hydroxyecdysone and juvenile hormone. Detr. Biol. 138, 214-224. Hiruma K. and Riddiford L. M. (1993) Molecular mechanism of cuticular melanization in the tobacco hornworm, Manduca sexta (L) (Lepidoptera: Sphingidae). Int. J. Insert Morphol. Embryol. 22, 103-I 17. Hiruma K., Riddiford L. M., Hopkins T. L. and Morgan T. D. (1985) Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco hornworm and their control by 20-hydroxyecdysone. Comp. Physiol. 155B, 659-669. Hori M., Hiruma K. and Riddiford L. M. (1984) Cuticular melanization in the Tobacco Hornworm larva. Insect Biochem. 14,267F274. Hopkins T. L., Morgan T. D., Aso Y. and Kramer K. J. (1982) N-b-alanyldopamine: A major role in insect cuticle tanning. Science 217, 364. Karlson P. and Sekeris S. E. (1962) N-Acetyldopamine as sclerotizing agents of the insect cuticle. Nuture 195, 364.366. Kayser H. (1985) Pigments. In: Comprehensive Insecr Physiology, Biochemistry and Pharmacology (Edited by Kerkut G. A. and Gilbert L. I.), Vol. 10. Pergamon Press. Oxford.

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Koch P. B. (1991) Precursors of pattern specific ommatin in red wing scales of the polyphenic butterfly Aruschniu fevuna L.: Haemolymph tryptophan and 3-hydroxy-kynurenine. Insect Biochem. 21,785-794. Koch P. B. (1992) Seasonal polyphenism in butterflies: A hormonally controlled phenomenon of pattern formation. ZooI. Jb. Physiol. 96, 227-240. Koch P. B. (1993) Production of [‘4C]-labeled 3-Hydroxy-Lkynurenine in a butterfly, Heliconius churitoniu L. (Heliconidae), and precursor studies in butterfly wing ommatins. Pigment Cell Res. 6, 8590. Koch P. B. (1994) Wings of the butterfly Precis coeniu synthesize dopamine melanin by selective enzyme activity of dopadecarboxylase. Nuiurwissenschuften 81, 3638. Kramer K. J. and Hopkins T. L. (1987) Tyrosine metabolism for insect cuticle tanning. Arch. Insect Biochem. Physiol, 6, 279-301. Kramer K. J., Morgan T. D., Hopkins T. L., Roseland C. R., Aso Y., Beeman R. W. and Lookhart G. L. (1984) Catecholamins and B-alanine in the red flour beetle Tribolium castuneum. Insect Biothem. 14, 2933298. Kraminsky G. P., Clark W. C., Estelle M. A., Gietz R. D., Sage B. A., O’Connor J. D. and Hodgetts R. B. (1980) Induction of translatable mRNA for dopa decarboxylase in Drosophila: An early response to ecdysterone. Proc. Nutl. Acud. Sci. USA 17, 41754179. Maisch A. (1985) Melanisierungsund Sklerotisierungssteuerung bei der morphologischen Farbanpassung der Puppen von Inachis io (L.). Thesis, Ulm, Germany. Maisch A. and Bilckmann D. (1987) The control of cuticular melanin and lutein incorporation in the morphological colour adaptation of a nymphalid pupa, Inachis io L. J. Insect Physiol. 33, 393402. McCamman M. W., McCamman R. E. and Lees G. J. (1972) Liquid cation exchange-a basis for sensitive radiometric assays

for aromatic amino acid decarboxylases. Anal. Biochem. 45, 242-252. Nagatsu T., Levitt M. and Udenfriend S. (1964) Tyrosine hydroxylase. J. biol. Chem 239, 2910-2917. Nijhout H. F. (1980a) Pattern formation on lepidopteran wings: Determination of an eyespot. Dev. Biol. 80, 267-274. Nijhout H. F. (1980b) Ontogeny of the color pattern on the wings of Precis coenia (Lepidoptera: Nymphalidae). Dev. Biol. 80, 2755288. Nijhout H. F. (1985) The developmental physiology of color patterns in Lepidoptera. Adv. Insect Physiol. 18, 181-247. Nijhout H. F. (1991) The Development and Evolution of Butterfly Wing Patterns. Smithsonian Institution Press, Washington and London. Nijhout H. F. and Koch P. B. (1991) The distribution of radiolabeled pigment precursors in the wing patterns of nymphalid butterflies J. Res. Tepid. 30, l-13. Roseland C. R., Kramer K. J. and Hopkins T. L. (1987) Cuticular strength and pigmentation of rust-red and black strains of Tribohum custuneum. Insect Biochem. 17, 591-596. Umebachi Y. (1990) Beta-alanine and pigmentation of insect cuticle. In Molting and Metamorphosis (Edited by Ohnishi E. and Ishizaki H.), Japan Scientific Press/Springer-Verlag, Tokyo/Berlin.

Acknowledgements-We thank Dr Kyioshi Hiruma (Seattle) for his advice in the DDC assay and Ute Moschler for her technical assistance. We are grateful to Professor Yoshishige Umebachi (Kanazawa) for his advice to study p-alanine in butterfly wings. We thank Professor D. Biickmann (Ulm) for his helpful discussions and Professor Paul M. Brakefield (Leiden) for corrections on the text. The work was partially supported by the LFSP Baden-Wtirttemberg “Molekulare Grundlagen der Zellularen Differenzierung”, project C4.